ES2647790T3 - Piperidyl-ethyl-pyrimidine substituted as an inhibitor of ghrelin O-acyl transferase - Google Patents

Abstract

A compound of formula ** Formula ** or a pharmaceutically acceptable salt thereof

Description

Piperidyl-ethyl-pyrimidine substituted as an inhibitor of ghrelin O-acyl transferase

The present invention relates to a compound useful for inhibiting ghrelin O-acyl transferase (GOAT), pharmaceutical compositions and compounds for use in methods for treating diseases related to GOAT activity 5.

GOAT belongs to the family of membrane-bound O-acyl transferase enzymes (MBOAT). This converts desacilghrelin (also known as deacylated ghrelin or UAG) into a biologically active form, acylghrelin (AG), by transferring a Ser3 fatty acid to the residue of desacilghrelin peptide. Acylghrelin has been shown to increase food intake and decrease adiposity in humans and rodents. The injection of AG in humans has also been shown to suppress glucose-induced insulin secretion. The elimination of the ghrelin gene has been shown to enhance the release of insulin to prevent or improve lactose intolerance in ob / ob mice with a high-fat diet.

The prevalence of obesity and diabetes accompanied by variable effectiveness and responses to current treatments for obesity and diabetes makes it necessary for there to be more treatment options for patients. It is believed that a GOAT inhibitor is a useful agent in the treatment of obesity. It is further believed that a GOAT inhibitor is also useful in reducing weight gain or weight recovery as a supplement to a diet and / or exercise, other therapeutic medicinal agents or procedures designed to reduce weight gain or Treat obesity. Similarly, a GOAT inhibitor may be useful in the treatment of type 2 diabetes, alone or in combination with other treatments for type 2 diabetes. WO2007079239 discloses bicyclic nitrogen compounds as modulators of the receptor of Ghrelin The

The present invention provides a compound that is a GOAT inhibitor. In particular, the present invention provides a compound of formula

image 1

or a pharmaceutically acceptable salt thereof.

The present invention further provides a compound that is a GOAT inhibitor of formula

image2

The present invention provides a pharmaceutical composition comprising a compound of the invention, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers, diluents or excipients. In a further embodiment, the present invention provides a pharmaceutical composition that

30 comprises a compound of the invention and one or more other therapeutic agents.

A further aspect of the present invention provides compounds for use in a method of weight gain reduction or weight recovery or treatment of type 2 or obesity diabetes comprising the administration of a compound of the present invention or a pharmaceutically salt. acceptable to a patient who needs it.

The present invention further provides a compound of the invention, or a pharmaceutically acceptable salt thereof, for use in therapy, in particular to reduce weight gain or weight recovery or treatment of type 2 diabetes or obesity. On the other hand, the present invention provides the use of a compound of the invention or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the reduction of weight gain or weight recovery or treatment of type 2 or diabetes. obesity.

The compound of the present invention is generally effective over a wide dosage range. For example, dosages per day are in the range of about 0.03 to

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approximately 150 mg / kg body weight. In some cases the dosage levels below the limit of the aforementioned interval may be more adequate, while in other cases even larger doses may be used while maintaining a favorable benefit / risk profile, and therefore, the interval The foregoing is not intended to limit the scope of the invention in any way. It will be understood that the amount of the compound administered will actually be determined by a physician, in view of the relevant circumstances, including the condition to be treated, the route of administration selected, the compound or actual compounds administered, age, weight and the response of the individual patient, as well as the severity of the patient's symptoms.

The term "treating" (or "treatment" or "treatment") as used herein refers to the containment, slowdown, arrest or reversal of the progression or severity of an existing symptom, condition or disorder.

As used herein, the term "reduction in weight gain" refers to the decrease in weight gain of a patient. The term "reduction in weight recovery" refers to the decrease in weight gain of a patient who experiences a rebound in weight after weight loss. Weight recovery may be due to a flanging effect followed by the cessation of weight loss achieved through diet, exercise, behavior modification or approved therapies. For no doubts, weight gain or weight recovery as used herein, it refers to weight gain or weight recovery induced by food intake or eating habits and does not refer to weight gain. Not related to food such as fluid accumulation, weight due to water retention, muscle mass or inflammation.

A compound of the present invention can react to form pharmaceutically acceptable salts. Pharmaceutically acceptable salts and the common methodology for preparing them are well known in the art. See, for example, P. Stahl, et al. Handbook of Pharmaceutical Salts: Properties, Selection and Use, 2nd Revised Edition (Wiley-VCH, 2011); YE. Berge, et al., "Pharmaceutical Salts," Journal of Pharmaceutical Sciences, vol. 66, No. 1, January 1977.

The person skilled in the art will appreciate that the compound of the invention or the pharmaceutically acceptable salt thereof, are comprised of a core containing at least one chiral center:

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Although the present invention contemplates all individual enantiomers, as well as mixtures of the enantiomers of said compounds that include racemates, the preferred compound of the invention is represented by the formula:

image4

or pharmaceutically acceptable salts thereof.

A compound of the present invention is preferably formulated as pharmaceutical compositions administered by a variety of routes. Such pharmaceutical compositions and processes for the preparation thereof are well known in the art. See, for example, Remington: The Science and Practice of Pharmacy (A. Gennaro, et al., Eds., 21st ed., Mack Publishing Co., 2005). More particularly preferred, a pharmaceutical composition comprising a compound of the invention represented by the formula

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or a pharmaceutically acceptable salt thereof and one or more pharmaceutically acceptable carriers or diluents.

Single enantiomers or diastereomers can be prepared starting with chiral reagents or by stereoselective or stereospecific synthetic techniques. Alternatively, single enantiomers or diastereomers can be isolated from mixtures by standard chromatographic or chiral crystallization techniques at any convenient point in the synthesis of compounds of the invention. Unique enantiomers or diastereomers of compounds of the invention are a preferred embodiment of the invention.

It is well known in the art that agents for the treatment of diabetes and / or obesity can be combined with other agents for the treatment of diabetes and / or obesity. The compound of the invention, or a pharmaceutically acceptable salt thereof, can be co-administered, simultaneously or sequentially, with another effective treatment (s) for diabetes or obesity. The compound of the invention, or a pharmaceutically acceptable salt thereof, can be administered alone or in combination with other effective treatment (s), simultaneously or sequentially, followed by approved medical procedures such as bariatric surgeries, for example. , gastric bypass surgery or adjustable gastric band procedures.

The present invention also encompasses all processes for the synthesis of the compound of the present invention.

Additionally, the intermediates described in the following schemes may contain a number of nitrogen protection groups. The variable protection group may be the same or different at each occurrence depending on the particular reaction conditions and the particular transformations to be performed. The protection and deprotection conditions are well known to the person skilled in the art. See. for example, Greene and Wuts, Protective Groups in Organic Synthesis, (T. Greene and P. Wuts, eds., 2nd ed. 1991).

Preparations and Examples

The following preparations and examples further illustrate the invention and represent the typical synthesis of the compound of the invention. Reagents and starting materials are readily available or can be easily synthesized by a person skilled in the art. It should be understood that the preparations and examples are presented below for illustrative and non-limiting purposes, and that various modifications can be made by a person skilled in the art.

The R or S configuration of the compound of the invention can be determined by standard techniques such as X-ray analysis and correlation with chiral HPLC retention time. The names of the following preparations and examples are made using the IUPAC name feature in MDL Accelrys® Draw version 4.0.NET.

As used herein, the following terms have the meanings indicated: "ACN" refers to acetonitrile; "BSA" refers to bovine serum albumin; "DCM" refers to dichloromethane; "DIPEA" refers to W, W-diisopropylethylamine; "DMF" refers to dimethylformamide; "DMSO" refers to dimethylsulfoxide; "EDTA" refers to ethylenediaminetetraacetic acid; "EtOAc" refers to ethyl acetate; "EtOH" refers to ethanol; "FBS" refers to fetal bovine serum; "HRP" refers to horseradish peroxidase; "IC50" refers to the concentration of an agent that produces 50% of the maximum response; "IPA" refers to isopropyl alcohol; "MeOH" refers to methanol; "MTBE" refers to methyl tert-butyl ether; "PBS" refers to phosphate buffered saline; "TA" refers to room temperature; "TEA" refers to triethylamine; "TFA" refers to trifluoroacetic acid; "Tr" refers to retention time; "THF" refers to tetrahydrofuran; and "TMB" refers to 3.3 ', 5.5 '-tetramethylbenzidine.

LC-MS conditions (low pH): column: Phenomenex Gemini NX C18 2.1 x 50 mm 3.0 m; gradient: 5-100% B in 3 min, then 100% B for 0.75 min column temperature: 50 ° C +/- 10 ° C; flow rate: 1 ml / min; Solvent A: deionized water with 0.1% formic acid; Solvent B: ACN with 0.1% formic acid.

Preparation 1

6-Chloro-2-methylpyrimidine-4-amine

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A 5 L metal pressure vessel (autoclave) is charged with 4,6-dichloro-2-methylpyrimidine (400 g, 2.45 mol) and ammonium hydroxide (2.8 l) at RT. The reaction is heated at 90 ° C for 5 h. The reaction mixture is cooled to RT and then the solid is filtered through a Büchner funnel. The filter cake was cleaned with water (200 ml) and hexane (200 ml) and then dried under vacuum to obtain the title compound as a white solid (290 g, 82%). LC-ES / MS m / z 144.0 (M + 1).

Preparation 2

6-Chloro-5-iodo-2-methylpyrimidine-4-amine

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6-Chloro-2-methylpyrimidin-4-amine (708 g, 4.93 mol) and MeOH (7.08 l) are combined in a 20 l round bottom flask equipped with a mechanical stirrer. It cools to 0-5 ° C. Iodine monochloride (4.806 kg, 29.6 mol) dissolved in MeOH (6 L) is added using an addition funnel over a period of 1 h. The reaction mixture is heated at RT and stirred at RT for 16 h. The reaction mixture is cooled to 0-5 ° C and sodium sulphite (46 L, 20% aqueous solution) is added. The resulting solid is filtered and washed with water (2 L) followed by hexane (3 L). The solid is dried under vacuum to provide the title compound as a white solid (1061 g, 80%). LC-ES / MS m / z 269.9 (M + 1).

Preparation 3

tert-Butyl 4- [2- (4-amino-6-chloro-2-methyl-pyrimidin-5-yl) ethynyl] piperidine-1-carboxylate

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6-Chloro-5-iodo-2-methyl-pyrimidin-4-amine (20 g, 74.22 mmol), 4-ethynyl-piperidine-1-carboxylic acid tert-butyl ester (18.64 g, 89) is dissolved , 06 mmol), and diisopropylamine (10.44 ml, 74.22 mmol) in THF (200 ml) in a 3-mouth flask. Alternatively, the flask with nitrogen is evacuated and charged 3 times. It adds

bis (triphenylphosphine) palladium (II) chloride (2.63 g, 3.71 mmol) and copper (I) iodine (0.713 g, 3.71 mmol) to the solution. The mixture is heated at 50 to 55 ° C for 16 h. The mixture is cooled to RT and more is added.

bis (triphenylphosphine) palladium (II) chloride (1.31 g, 1.86 mmol), copper (I) iodine (0.356 g, 1.86 mmol) and 4-ethynyl-piperidine-1- carboxylic acid tert-butyl ester (1.55 g, 7.42 mmol). The mixture is heated at 60 ° C for 3.5 h. The mixture is cooled to RT and concentrated under reduced pressure. The material is diluted with DCM (300 ml) and lacquered with saturated aqueous ammonium chloride solution (100 ml), water (100 ml) and saturated aqueous sodium chloride (100 ml). The solution is dried over MgSO4, filtered and concentrated under reduced pressure. The residue is purified using silica gel chromatography (800 g of silica gel column) eluting with 20% to 100% EtOAc in hexanes. The purified fractions are concentrated to provide the title compound as a pale orange powder (22.6 g, 86%). LC-ES / MS m / z (35Cl / 37Cl) 351.2 / 353.1 (M + 1).

Preparation 4

tert-Butyl 4- [2- (4-amino-6-chloro-2-methyl-pyrimidin-5-yl) ethyl] piperidine-1-carboxylate

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Combine tert-butyl 4- [2- (4-amino-6-chloro-2-methyl-pyrimidin-5-yl) ethynyl] piperidine-1-carboxylate (4.30 g, 12.26 mmol) and platinum ( IV) oxide (0.139 g, 0.61 mmol) in EtOH (81 ml) and EtOAc (40 ml). The flask is alternatively evacuated and charged with hydrogen using a hydrogen balloon and stirred at RT for 8 h. The reaction is carefully controlled to avoid a potential derivative product that results from the removal of the chloride in the molecule. It should be noted that the product is more soluble in the solvent mixture than the starting alkyne. The mixture is filtered through diatomaceous earth, rinsing with MeOH. The solution is concentrated under reduced pressure. Silica, 1-propanethiol (4 g, load = 1.28 mmol / g, SILIABOND® Thiol of SILiCyCLE®) (to remove residual palladium from the previous coupling reaction) and EtOAc (300 ml) are added in a flask. The material is stirred at RT for 3 days. The solids are filtered and the filtrate is concentrated under reduced pressure. The hydrogenation is repeated on the resulting residue as follows. The flask containing the residue is charged with platinum (IV) oxide (0.139 g, 0.61 mmol), EtOH (81 ml) and EtOAc (40 ml). Alternatively, the flask is evacuated and charged with hydrogen using a hydrogen balloon and stirred at RT for 8 h. It is filtered through diatomaceous earth, rinsing with MeOH and the filtrate is concentrated under reduced pressure. The hydrogenation is repeated on the resulting residue as follows. The flask containing the residue is charged with platinum (lV) oxide (0.139 g, 0.61 mmol), EtOH (81 ml) and EtOAc (40 ml). Alternatively, the flask is evacuated and charged with hydrogen using a hydrogen balloon and stirred at RT for 8 h. It is filtered through diatomaceous earth, rinsing with MeOH. The filtrate is concentrated under reduced pressure on silica gel (20 g). The material is purified using silica gel chromatography eluting with 70% to 100% ErOAc in hexanes (120 g column). The purified fractions are combined and concentrated under reduced pressure. The residue is diluted with DCM and hexanes and concentrated under reduced pressure three times. The material is placed under vacuum to provide the title compound as a white solid (3.20 g, 73%). LC-ES / MS m / z (35Cl / 37Cl) 355.2 / 357.2 (M + 1).

Preparation 5

6-Chloro-2-methyl-5- [2- (4-piperidyl) ethyl] pyrimidine-4-amine, hydrochloride salt

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Ferc-Butyl 4- [2- (4-amino-6-chloro-2-methyl-pyrimidin-5-yl) ethyl] piperidine-1-carboxylate (29.00 g, 81.72 mmol) is dissolved in 1, 4-dioxane (145 ml) and 4M of hydrogen chloride in 1,4-dioxane (204.2 ml, 817.1 mmol) are added. Stir for 18 h at RT. The mixture is concentrated under reduced pressure, suspended in diethyl ether (250 ml), filtered and dried the resulting solid in vacuo to give the title compound as a crude white solid (29 g). LC-ES / MS m / z (35Cl / 37Cl) 255.2 / 257.2 (M + 1).

Alternative route for 6-chloro-2-methyl-5- [2- (4-piperidyl) ethyl] pyrimidin-4-amine (Preparations 6-10)

Preparation 6

tert-Butyl 4- (2-methylsulfonyloxyethyl) piperidine-1-carboxylate

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Tert-butyl 4- (2-hydroxyethyl) piperidine-1-carboxylate (4,720 kg, 20.6 mol) is combined with DCM (40 l) and TEA (3020 ml, 21.63 mol) in a 50 l reactor with An atmosphere of nitrogen. The solution is cooled to about 0 ° C and methyl sulfonyl chloride (2,478 kg, 21.63 mol) in DCM (5 L) is slowly added while maintaining the reaction temperature below 10 ° C. After the addition is complete, stir the mixture for 18 h at 15 ° C at the time when TLC (1: 1, hexane: EtOAc) shows no remaining starting material. The mixture is washed with water (30 l) and the phases are allowed to separate. The organic phase is concentrated to a solid. Be

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The solid is suspended in MTBE (6 L), collected by filtration and dried in a vacuum oven at 50 ° C to provide the title compound as a white solid (5.67 kg, 91%).

Preparation 7

tert-Butyl 4- (3-cyano-4-ethoxy-4-oxo-butyl) piperidine-1-carboxylate

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Tert-butyl 4- (2-methylsulfonyloxyethyl) piperidine-1-carboxylate (5.26 kg, 17.1 mol), ethyl cyanoacetate (7 kg, 62 mol), 21% sodium ethoxide in EtOH (8, 25 l, 24.75 mol) and EtOH (35 l) in a 50 l reactor with a nitrogen atmosphere. The mixture is heated at 35-40 ° C for 18 h at the time the NMR analysis shows 20% of the remaining mesylate. The mixture is continued to be heated at 35-40 ° C for 24 h at the time when the NMR analysis shows only a small amount of mesylate. The reaction is slowly cooled to RT and glacial acetic acid (1422 ml, 22.68 mol) is added for 30 min. The mixture is concentrated using vacuum distillation and then partition between water (25 L) and EtOAc (35 L). The layers are separated and the aqueous phase is mixed with EtOAc (10 L). The organic portions are combined, washed with brine (15 L) and concentrated in a red oil. The oil is purified using silica gel chromatography (75 kg of silica gel, 65-250 mesh), eluting with hexane (40 L) and then 20% EtOAc / hexane to provide a fraction (7.8 kg) that is, 30% ethyl cyanoacetate / 70% of the desired product. The material is concentrated by distillation of rinsed sheet at 100 ° C and 210 mtorr under vacuum, the non-volatile fraction is collected to provide the title compound (4.54 kg, 82%).

Preparation 8

tert-Butyl 4- [2- (4-amino-6-hydroxy-2-methyl-pyrimidin-5-yl) ethyl] piperidine-1-carboxylate

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Combine tert-butyl 4- (3-cyano-4-ethoxy-4-oxo-butyl) piperidine-1-carboxylate (2.18 kg, 6.73 mol), acetamidine hydrochloride (1.27 kg, 13, 46 mol, 95% test) [Note: Acetamidine hydrochloride is pre-dried by suspending two in toluene (10 l) and then extracting until vacuum drying], 21% sodium ethoxide in EtOH (6, 73 l, 20.19 mol) and EtOH (10 l) in a 22 l reactor with a nitrogen atmosphere. The reaction is heated at reflux for 42 h. The reaction is adjusted to approximately pH = 5 with glacial acetic acid (1.2 L, 20.86 mol). EtOH is removed by vacuum distillation on a rotary evaporator. The resulting solids are suspended in water (6 L) and then the solids are collected by filtration, washed with additional water (2 L). The solids are dried in a vacuum oven at 50 ° C to provide the title compound (1.72 kg, 76%). 1H NMR (500 MHz, CD3OD) or 4.83 (s, 3H; 4.05 (d, 2H), 2.75 (bs, 2H), 2.39 (m, 2H), 2.22 (s, 3H), 1.78 (d, 2H), 1.43 (m, 1H) 1.42 (s, 9H), 1.40 (m, 2H), 1.38 (m, 2H). procedure, essentially as described, using tert-butyl 4- (3-cyano-4-ethoxy-4-oxo-butyl) piperidine-1-carboxylate (2.36 kg) to obtain the title compound (2.01 kg , 82%).

Preparation 9

6-Amino-2-methyl-5- [2- (4-piperidyl) ethyl] pyrimidin-4-ol, dihydrochloride salt

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Combine tert-butyl 4- [2- (4-amino-6-hydroxy-2-methyl-pyrimidin-5-yl) ethyl] piperidine-1-carboxylate (3.73 kg, 11.09 mol) and IPA in a 50 l reactor with a nitrogen atmosphere. 12 N hydrochloric acid (3.05 L, 36.6 mol) are added with stirring for 3 h. The mixture is heated at 50 ° C for 16 h, forming a thick suspension. At that time the NMR analysis does not show any remaining BOC group. The reaction mixture is cooled to 20-25 ° C, diluted with THF (12 L). The solids are collected by filtration. Filtration is slow and can take about 24 hours to complete. It is cleaned with IPA (2 x 10 l). The material is dried in a vacuum oven at 50 ° C to provide the title compound (3,117 kg, 91%).

Preparation 10

6-Chloro-2-methyl-5- [2- (4-piperidyl) ethyl] pyrimidine-4-amine, dihydrochloride salt

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Combine 6-amino-2-methyl-5- [2- (4-piperidyl) ethyl] pyrimidin-4-ol, hydrochloride salt (3,167kg, 10.2 mol) and phosphorus oxychloride (30 l, 300 mol ) with a nitrogen atmosphere in a 50 l reactor that is vented through a caustic scrubber (10% NaOH). 85% phosphoric acid (1 L, 8.5 mol) is added to the stirred suspension. The mixture is heated slowly using a reactor jacket temperature of 75 ° C. At approximately 55 ° C, when washing occurs, the washer is cooled in a water ice bath. When the gas release is slowed, the mixture is heated at 90-95 ° C for 50 h at the time when the TLC and NMR analysis shows that the reaction is complete. The mass of excess phosphorus oxychloride is removed by vacuum distillation (22 in Hg under vacuum and 60 ° C at internal temperature) to recover 20 l in the distillate. The mixture is cooled to 20-25 ° C and then diluted with toluene (10 L). The mixture is further cooled to <15 ° C and slowly quenched with water (1 L) maintaining the temperature <30 ° C. After the addition of toluene the mixture becomes very expected and complicated to stir with the product phase remaining at the bottom of the reactor as a viscous semi-solid. The agitator blades are raised to avoid getting any mixture. EtOH (10 L) is added to the mixture and stirred for approximately 18 h at 25 ° C. At this point, the viscous semi-solids are not yet fully digested, but softened so that they can be analyzed manually. Stir vigorously for another 4-5 h to complete digestion. The resulting suspension is cooled to 5-10 ° C and the solids are collected by filtration, washing with MTBE (8 L) to obtain a wet product (approximately 6 kg).

The crude product is recrystallized by dissolving the wet cake in MeOH (35 L) at 40 ° C. The solution is concentrated by vacuum distillation, removing 15 l of solvent. IPA (35 L) is added and the suspension is concentrated by vacuum distillation to remove 13 L of solvent. The suspension is cooled to 5-10 ° C and then the solids are filtered, cleaning with IPA (2 L) and then MTBE (8 L). The solids are dried in a vacuum oven at 55 ° C to achieve the title compound as a bleached solid (2.77 kg, 82%). Analytical calculated for C12H21ClaN4: C, 43.98; H, 6.46; Cl, 32.46; N, 17.10. Found: C, 43.63; H, 6.53; Cl, 32.16; N, 16.86.

Preparation 11

tertc-Butyl W - [(1S) -2- [4- [2- (4-amino-6-chloro-2-methyl-pyrimidin-5-yl) ethyl] -1-piperidyl] -1-methyl-2 -oxo-ethyl] carbamate

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6-Chloro-2-methyl-5- [2- (4-piperidyl) ethyl] pyrimidin-4-amine hydrochloride (12.50 g, 42.92 mmol), diisopropylethylamine are added in each of two round bottom flasks (22.46 ml, 128.7 mmol) and dMf (100 ml). The two mixtures are cooled in a cold water bath and stirred for 5 min. To each of the mixtures [dimethylamino (triazolo [4,5-b] pyridin-3-yloxy) methylene] -dimethyl-ammonium hexafluorophosphate (17.95 g, 47.21 mmol) and (2S) -2- (ferc-butoxycarbonylamino) propanoic acid (8.93 g, 47.21 mmol) in one portion. The mixtures are stirred at RT for 90 min. The mixtures are poured into separate separating funnels together with water (300 ml) and EtOAc (400 ml), stirring and partitioning. The aqueous layers are extracted with EtOAc (3 x 300 ml), the respective organic layers are washed with water (4 x 250 ml), saturated aqueous NaCl (200 ml) and dried over MgSO4, filtered and concentrated under reduced pressure. . The combined materials are purified by chromatography on silica gel eluting with 70% to 100% EtOAc in hexanes. The purified fractions are combined and concentrated under reduced pressure. Be

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Dilute the residue with EtOAc (500 ml) and wash with saturated aqueous NH4CI (100 ml), saturated aqueous NaHCO3 (100 ml), water (100 ml) and saturated aqueous NaCl (100 ml). The organics are dried over MgSO4, filtered and concentrated under reduced pressure to give the title compound as a white solid (28.00 g, 76%). LC-ES / MS m / z (35Cl / 37Cl) 426.2 / 428.2 (M + 1).

Preparation 12

(2S) -2-Amino-1- [4- [2- (4-amino-6-chloro-2-methyl-pyrimidin-5-yl) ethyl] -1-piperidyl] propan-1-one

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Ferc-butyl W - [(1S) -2- [4- [2- (4-amino-6-chloro-2-methyl-pyrimidin-5-yl) ethyl] -1-piperidyl] -1-methyl is dissolved -2-oxo-

ethyl] carbamate (15.78 g, 37.05 mmol) in DCM (185 ml), TFA (185 ml) is added dropwise over 3 min, and stirred overnight. The reaction is analyzed by LCMS (low pH) to show complete conversion. MeOH (400 ml) is slowly added due to the exothermic mixture. Three SCX columns (50 g) are previously washed with water (20 ml) and then MeOH (20 ml). The reaction mixture is divided into three equal portions and loaded equally on the SCX columns. Each column is washed with water (40 ml) and MeOH (40 ml) collecting the washings in a vacuum flask. The desired material of the SCX columns is eluted with 2 N ammonium in MeOH (60 ml) in a clean container. The solutions containing the product are combined, concentrated under reduced pressure, azeotropically mixed with DCM-hexanes (1: 1), three times and placed under vacuum to obtain the title of the compound as a white foam (10, 29 g, 84%). LC-ES / MS m / z (35Cl / 37Cl) 326.2 / 328.2 (M + 1).

Preparation 13

(2S) -2-Amino-1- [4- [2- (4-amino-6-chloro-2-methyl-pyrimidin-5-yl) ethyl] -1-piperidyl] propan-1-one salt hydrochloride

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Ferc-butyl N - [(1S) -2- [4- [2- (4-amino-6-chloro-2-methyl-pyrimidin-5-yl) ethyl] -1-piperidyl] -1-methyl is added -2-oxoethyl] carbamate (28.75 g, 67.49 mmol) to 1,4-dioxane (143.7 ml). Then 4M of hydrogen chloride in 1,4-dioxane (168.7 ml, 674.9 mmol) is added and stirred for 10 min. MeOH (20 ml) is added and the mixture is stirred for 3 h with vigorous stirring. The mixture is concentrated under reduced pressure, the solids are diluted with diethyl ether (200 ml) and stirred overnight. The material is filtered, rinsed with diethyl ether (2 x 25 ml). The material is dried through suction for 15 min, then placed in vacuo for 1 h at 45 ° C to obtain the title compound as a crude white powder (27.7 g). LC-ES / MS m / z (35Cl / 37Cl) 326.1 / 328.2 (M + 1).

Preparation 14

(2S) -2-Amino-1- [4- [2- (4-amino-6-chloro-2-methyl-pyrimidin-5-yl) ethyl] -1-piperidyl] propan-1-one, salt of dihydrochloride

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IPA (154 ml) is heated to 50 ° C and acetyl chloride (19.3 ml, 271 mmol) is added slowly due to an exothermic reaction. The reaction is stirred at 50 ° C for 10 minutes and then ferc-butyl W - [(1S) -2- [4- [2- (4- amino-6-chloro-2-methyl-pyrimidin-5-) is added il) ethyl] -1-piperid1] -1-methyl-2-oxo-ethyl] carbamate (21.0 g, 45.3 mmol). Stir the

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reaction for 2 h controlling through LCMS (low pH). The reaction is cooled to RT and diethyl ether (386 ml) is added. The suspension is stirred for 15 min. The solids are filtered, washed with diethyl ether (2 x 50 ml) in an energetic manner since the material is hydroscopic. The material is filtered and dried through filtration for 1 min, then in a vacuum drying oven at 50 ° C overnight to provide the title compound as a white powder (18.4 g). LCES / MS m / z (35Cl / 37Cl) 326.1 / 328.2 (M + 1). The counter-ion analysis by ion chromatography is consistent with the dihydrochloride salt.

Example 1

N - [(1S) -2- [4- [2- (4-Amino-6-chloro-2-methyl-pyrimidin-5-yl) ethyl] -1-piperidyl] -1-methyl-2-oxo- ethyl] cyclopropanecarboxamide

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1-Hydroxy-7-azabenzotriazole (281 mg, 2.03 mmol) and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (430 mg, 2.21 mmol) are added to a suspension of cyclopropanecarboxylic acid (161 pl, 2.03 mmol), (2S) -2-amino-1- [4- [2- (4-amino-6-chloro-2-methyl-pyrimidin-5-yl) ethyl] -1-piperidyl] propan- 1-one (600 mg, 1.84 mmol) in anhydrous THF (12 ml). TEA (770 pl, 5.52 mmol) is then added and the resulting mixture is stirred at RT for 12 h. The reaction mixture is diluted with EtOAc (10 ml), filtered over diatomaceous earth and the filtrate is concentrated in vacuo. The resulting residue is purified by mass-guided HPLC reverse phase chromatography (Agilent® 1200 LCMS and 75 x 30 mm MSD mass spectrometer Phenomenex Gemini-NX®, 5 p particle size column with a 10 x 20 mm protector , 12-46% ACN in 10 mM aqueous ammonium bicarbonate solution, pH 10, gradient over 9 min) to obtain the title compound as a colorless glass (572 mg, 78%). LCESMs m / z (35Cl / 37Cl) 394.2 / 396.2 (M + H).

Alternative procedure with 1-propanophosphonic anhydride

A mixture of (2S) -2-amino-1- [4- [2- (4-amino-6-chloro-2-methyl-pyrimidin-5-yl) ethyl] -1-piperidyl] propan-1 is treated -one, hydrochloride salt (107.4 g, 183.15 mmol) in DcM (584 ml) with DIPEA (128 ml, 732.59 mmol). The reaction mixture is cooled to 0 ° C in an ice bath and cyclopropanecarboxylic acid (21.8 ml, 274.72 mmol) is added. A solution of 1-propanophosphonic anhydride (50% solution in EtOAc, 174.8 g, 274.72 mmol) is then added followed by additional DIPEA (40 ml) to convert the basic reaction mixture. Shake at RT overnight. The reaction mixture is washed with water (1 L) and the layers are separated. The organic portion is dried over MgSO4, concentrated in vacuo to obtain the crude product as an off-white foam. The material is purified by chromatography on silica gel (800 g, 0-10% MeOH in EtOAc). The appropriate fractions were combined with another batch product (51 g) obtained by monitoring essentially the same procedure using (2S) -2-amino-1- [4- [2- (4-amino-6-chloro-2- methyl-pyrimidin-5-yl) ethyl] -1-piperidyl] propan-1-one, dichlorohydrochloride salt (77.9 g, 150.42 mmol). The combined batches were concentrated in vacuo to obtain a white solid (114 g). The product began to crystallize during concentration. The material is triturated with hot EtOAc (200 ml) and filtered to obtain the title compound (111 g, 84%). LC-ES / MS m / z (35Cl / 37Cl) 394.0 / 396.0 (M + 1). Chiral analysis (OD-H column, 25% MeOH / iPrNH2, 5.0 ml / min, 100 bar, 35 ° C, 220 nm)> 99.9% ee; Rt = 1.15 min.

GOAT is the main enzyme that converts UAG into AG. For reviews on the role of GOAT and ghrelin see: Kristy M. Heppner et al., The ghrelin O-acyltransferase-ghrelin system: a novel regulator of glucose metabolism, Current Opinion in Endocrinology, Diabetes & Obesity 2011, 18:50 -55; Phillip A. Cole et al., Glucose and Weight Control in Mice with a Designed Ghrelin OAcyltransferase Inhibitor, Science. December 17, 2010; 330 (6011): 1689-1692. doi: 10.1126 / science.1196154, Matthias H. Tschop et al., Gastric O-acyl transferase activates hunger signal to the brain, Proc Natl Acad Sci U S A. April 29, 2008; 105 (17): 6213-6214, and Jesus Gutierrez, et al., Ghrelin octanoylation mediated by an orphan lipid transferase, Proc Natl Acad Sci U S A., April 29, 2008, 105 (17): 6320-6325.

The role of GOAT is supported by the phenotypes observed in mice devoid of the GOAT gene. Therefore, GOAT inhibition is expected to decrease the circulation of AG and increase the circulation of UAG. Therefore, the ratio of AG to total ghrelin (UAG + AG) is reduced after the inhibited treatment of GOAT.

In vitro cell-free human GOAT enzyme assay

The human GOAT gene (reference number: NM_001100916) is subcloned into the baculoviral expression vector pAN51. The baculovirus raw material is prepared following the Bac-a-Bac protocol provided by the

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Vendor, Invitrogen, California, USA Five millimeters of human GOAT baculoviral raw material is added to 500 ml of Sf9 cells in a HyQ SFX-Insect ™ medium (HyClone catalog number SH30278.02) at a density of 1 x 106 cells per millimeter in an Erlenmeyer flask of 2 l. The flask with Sf9 cells infected with the human GOAT gene is placed on a plate shaker at 120 rpm at 28 ° C for 48 h. After 48 h of incubation, the cells are centrifuged at 1,000xg for 10 min at 4 ° C. The cell pellets are collected and stored at -80 ° C in a freezer until they are ready for an additional procedure.

Preparation of GOAT enzyme microsomal membrane for enzymatic assay:

One gram of cell suspensions is suspended in a 9 ml refrigerated homogenization buffer (50 mM Tris-HCl, 250 mM sucrose, adjusted to pH 7.5 and sterile filtered through a Milipore filter of 0 , 2 pm). The cell suspension is transferred to a Dounce glass homogenizer. The cell suspensions were homogenized with 40 impulses on ice. The homogenate is centrifuged at 3,000 rpm in a Beckman rotating basket rotor at 4 ° C for 10 min to remove unbroken cells. The supernatant is collected and centrifuged at 40,000 xg for 1 h at 4 ° C. The resulting membrane suspension is suspended in the homogenization buffer using a Douce glass homogenizer and stored at -20 ° C in the freezer for testing. For long-term storage of the membrane preparation of the human GOAT enzyme, the suspended membrane is stored in a freezer at -80 ° C.

Enzyme assay protocol of human GOAT:

Test compounds are prepared in DMSO to achieve a 0.2 mM raw material solution. The raw material solution is serially diluted in DMS to obtain a ten-point curve with final compound concentrations ranging from 10 pM to 0.5 nM in a 96-well round bottom plate. The enzyme and substrate solutions are prepared in an assay buffer (0.02% Tween ™ -20 in 50 mM Tris, pH 7.5 / 250 mM sucrose / 1 mg / ml BSA / 10 mM EDTA). The diluted compound (1 pl) is added to each well of row A to N of a low protein junction corresponding to a 384 well plate. A mixture of human GOAT substrate (10 pl), consisting of human desacylchhrelin-biotin (CPC Scientific Inc., 6.0 pM final), octanoyl-CoA (Sigma, 60 pM final) and an AG-specific antibody are added. (WO 2006/091381) (1.0 pg / ml final), to the compounds. The GOAT-His / sf9 enzyme preparation, which has been prepared in an assay buffer (9 pl), is added to each well of the plate containing the substrate and the test compounds resulting in a final concentration of 0, 01 pg / ml to start the reaction. The mixture is incubated for 1 h at RT on a gently rotating oscillator. 4 M guanidine hydrochloride (20 pl) is added to the wells, mixed and incubated for 3 h to stop the reaction.

ELISA plates (STREPTAVIDIN SPECTRAPLATE ™ 384, Perkin Elmer) are prepared by blocking with a 2% heat-deactivated FBS in PBS (40 pl) (Invitrogen) blocking buffer for 3 h. The blocking buffer of the ELISA plate is aspirated and blocking buffer (23 pl) is added to columns 1-24, rows A-N. Rows O and P are reserved for the standard acylghrelin curve. It is added to the reaction mixture (2 pl) to the ELISA plates. A standard 10-point curve (biotin-labeled ocatanoyl-ghrelin is prepared by 2X serial dilution in the blocking buffer containing 0.2M starting guanidine hydrochloride at 2.5 pM. The reaction mixture is incubated or AG standard labeled with biotin on the ELISA plate overnight at 4 ° C. The next day, the 3x plate is washed with wash buffer (0.1% Tween ™ -20 / PBS, 100 pl per well in each cycle of washing) The specific antibody AG (WO 2006/091381) (25 pl of 0.5 pg / ml in blocking buffer) is added to each well and incubated at RT for 1 h. The 3x plate is washed with the wash buffer, similar to the previous step G-HRP protein (25 pl) (Southern Biotech) diluted 3,000x in blocking buffer is added and incubated 1 h at RT The last 3x is washed with a wash buffer , as in the previous steps TMB reagent (25 pl) (Kirkegaard & Perry Laboratories, Inc.) is added to each well and allowed to develop for 20 min and stop with 1 M phosphoric acid (25 pl per well). Related plates at 450 nm using an Envision Multilabel plate reader. Ag levels are calculated against an adjusted standard curve and the percentage of inhibition is calculated. The 10-point inhibition curve is represented and adjusted with a four-parameter logistic equation to obtain IC50 values using Activity Base (see. 7.3.2.1).

Following a protocol essentially as described above, the compound of Example 1 shows an IC50 of approximately 192 nM ± 73, (n = 5). The data demonstrates that the compound of Example 1 inhibits the activity of the purified GOAT enzyme in vitro.

Comparing the change in the ratio of AC to total ghrelin in the treated group of the compound and that of the treated vehicle group reflects the degree of inhibition of GOAT enzyme in vivo, due to the dynamic procedure of UAG to AG using the enzyme of the GOAT. In vivo pharmacodynamic studies herein, the levels of AG and UAG in plasma and stomach in the treated vehicle and compound groups are measured by ELISA specifically with respect to these two analytes. The total ghrelin level of each sample is computed as the sum of AG and UAG for these ELISA measurements. The ratio of AG to total ghrelin is defined by the level of aG in each sample divided by the total level of ghrelin in the same sample. The levels of AG, UAG and ratio of AG to total ghrelin in the treated vehicle groups are computed and set as 100%. The relative change of these parameters in the group of the treated compound is then computed to determine the efficacy of the test compound.

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IDB 3-day dose-dependent in vivo study for the GOAT inhibitor:

Animals and treatment:

Male C57BL / 6 mice were purchased from Harían (Indianapolis, IN) 9 weeks old. The mice were housed individually in a temperature controlled facility (24 ° C) with a 12 h light / dark cycle (lights in 2,200 h) and free access to a standard rodent food (2014 diet, Harlan) and water is allowed. Typically, mice are used when they are 10-13 weeks old at the time of the study. On day 0 of the experiment, mice are randomly placed in treatment groups (N = 7 / group) so that each group has similar average body weights. On day 1 and 2, the animals are treated with a vehicle (1% hydroxyethyl cellulose, 0.25% Tween 80, 0.05% antifoam) or test compound prepared in a vehicle as a multi-dose suspension by mouth tube at 7 am and 7 pm. On day 3, the animals are fasted, transferred to clean cages and dosed with the vehicle or the test compound again at 8 am by oral probe. That same day at 1 pm, animals are sacrificed by decapitation to collect blood. For details on blood collection and plasma treatment see Plasma collection and ghrelin extraction from plasma in the section below.

Blood collection:

Approximately 600 μl of blood is collected in a previously weighed EDTA tube containing 600 μl (defined as Vconservant) of freshly prepared preservative (4 mM PEFABLOC® [4- (2-aminoethyl) benzenesulfonyl fluoride hydrochloride], 72 mM NaCl, 58 mM NaF, 0.032 N hydrochloric acid, pH 3.0) and mix immediately. The tube is weighed again and kept on ice. To accurately determine the exact blood volume of each sample using this blood collection procedure, the blood weight of each mouse is computed using the following equation:

Blood weight = (Weight of the tube containing blood + preservative) - (Weight of the tube containing

preservative)

Blood volume (Vsangre) = (Blood weight) / 1.06

Note, the density of rodent blood is assumed to be 1.06 g / ml.

15 minutes after blood collection, the samples are centrifuged at 5,000 rpm at 4 ° C for 8 min. Plasma (650 pl) is removed to a 5 ml glass tube containing 1 N hydrochloric acid (65pl), mixed and kept on ice.

Ghrelin extraction by SEP-PAK® column:

AG and UAG are removed from the plasma using a SEP-PAK®_C18 column to remove the interference before performing the ELISA. Solid phase extraction of AG and UAG peptides by SEP-PAK®_C18 columns can be performed on a vacuum manifold (Waters Corp) or using a perisltic pump. The SEP-PAK® column extraction procedure from the sample is applied independently to the plasma sample obtained from each individual reaton. The general extraction protocol is described below.

All solutions used for the complete SEP-PAK® column extraction protocol must be in a cold ice condition. Wet sEp-PAK® columns (WAT054960, Waters Corp, Milford MA) with 99.9% ACN / 0.1% TFA (1 ml of 100 ml solution of ACN / 0.1 ml of TFA). Pressure is applied to adjust the flow rate to approximately 1 ml / min to remove the liquid from the column bed but not allow the column to dry at any point. Once the liquid is removed from the column, the pressure is stopped. The columns are equilibrated with 3% ACN / 0.1% TFA (1 ml of 97 ml of water, 3 ml of ACN, 0.1 ml of TFA). Pressure is applied to adjust the flow rate to approximately 1 ml / min to remove the liquid from the column bed, but not allow the column to dry. Approximately 650 pl of acidified plasma (defined as Vpiasma added to column) are diluted to 1.4 ml of ice cold 0.1% TFA. All diluted acidified plasma from the previous stage is loaded onto the columns. Pressure is applied to adjust the flow rate to approximately 0.5 ml / min to allow the sample to pass through the column and the ghrelin peptides are absorbed on the column resin. The column is not allowed to dry. Wash with 3% ACN / 0.1% TFA (0.9 ml of 97 ml of water, 3 ml of ACN, 0.1 ml of TFA). Pressure is applied to adjust the flow rate to approximately 1ml / min to remove the liquid from the column bed but the column is not allowed to dry. Repeat washing two or more times. It is eluted with 60% ACN / 0.1% TFA (1 ml of 40 ml of water, 60 ml of ACN, 0.1 ml of TFA). A collection tube is placed below each column, pressure is applied to adjust the flow rate to approximately 0.5 ml / min to push the liquid through the column and collect the eluent into the collection tube. Samples are frozen on dry ice immediately. The samples are lyophilized in a Speed-vac (Model # SC110A, Savant) and stored at -20 ° C until the ELISA is performed.

ELISA test for ghrelin:

MULTI-ARRAY® MSD® 96-well plates are coated (Meso Scale Discovery, Gaithersberg, MD, Catalog #

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L15XA-3) with 100 µl of 1 jg / ml of an antibody (WO 2005/026211 and WO2006 / 019577) that recognizes the middle domain of both acyl and deacylated forms of ghrelin in PBS (Invitrogen). The sides of the plates are tapered to ensure a well coating, sealed with an adhesive plate sealer and incubated overnight at RT. The contents are discarded and Blocker ™ Casein in PBS (25 jl) (Thermo Scientific, Rockford, IL, Catalog # 37528) is added to each well. The plates are resealed and placed on a plate shaker at RT for 1 h.

Lyophilized preserved plasma samples are reconstituted from SEP-PAK® C18 column extraction in Blocker ™ Casein in PBS (400 pt each sample, this volume is defined as Vreconstitution), mixed well with a vortex mixer and incubate on ice for 45-60 min. The contents of the plates are discarded and samples of reconstituted plasma at 25 ml are added to each well. Standard curves of acylghrelin and dehydrated ghrelin with 8,000 pg / ml are prepared and serial dilutions 1: 4 are made for 8 total concentrations. The standards prepared in duplicate are added to the plates blocked with 25 ml in each well. The plates are sealed and incubated at RT on a plate shaker for 2 h.

Plaque contents are discarded and washed three times with PBS including 0.1% Tween ™ 20 (150 pl) (PBS-T). The acylghrelin specific antibody (WO 2006/091381) or the deacylated ghrelin specific antibody (WO 2006/055347) labeled with MSD SULFO-TAG ™ (Meso Scale Discovery) are diluted to 0.05 jg / ml at 0, 2 x Blocker Casein containing 0.05% Tween ™ 20, named secondary antibody solution. The final wash is removed and the secondary antibody solution (25 µl is added to each well) that specifically recognizes AG or UAG. The plates are released and incubated for 1 h at RT on a plate shaker before finally washing 3 x again with PBS-T (150 ml / well).

The final wash is discarded and replaced with 1 x MSD buffer and reading (150 jl / well). The electroquemiluminescent signal generated by activating the limit of the MSD SULFO-TAG ™ marker on the plates is read using the MSD® SECTOR® Imager 6000 (Meso Scale Discovery). Acylghrelin or deacylated ghrelin concentrations are calculated based on the respective standard curve generated by the MSD® software. The actual plasma concentration for each sample is determined by multiplying the level of acylghrelin or deacylated ghrelin measured by a dilution factor. The dilution factor for each plast sample is computed with the following equation.

image21

Results:

Administration of the compound of Example 2 for 3 days increases the Ag plasma by -1%, 5%, 45% and 48%, and the UAG increases by 2.24, 2.82, 2.53 and 2.89 times, respectively at 0.3, 1, 3 and 10 mg / kg (results tabulated below). Administration at 0.3, 1, 3 and 10 mg / kg results in 39, 54, 71 and 77% reduction respectively in AG with respect to the total ghrelin ratio when compared to control animals treated with vehicle. These results demonstrate that the compound of Example 1 suppresses the production of AG and elevates the circulating UAG, as shown in the mouse removed from GOAT, in vivo.

 Treatment

 AG (% control) UAG (% vehicle control) AG / Ghrelin-total (% vehicle control)

 Vehicle

 100 100 100

 0.3 mg / kg

 101 ± 25 224 ± 35 61 ± 4

 1 mg / kg

 95 ± 18 282 ± 45 46 ± 11

 3 mg / kg

 55 ± 6 253 ± 31 29 ± 4

 10 mg / kg

 52 ± 10 289 ± 44 23 ± 4

Claims (7)

1. A compound of formula image 1 or a pharmaceutically acceptable salt thereof 2. The compound of claim 1 of formula image2 or a pharmaceutically acceptable salt thereof 3. The compound of any of claims 1 or 2 of formula image3 A pharmaceutical composition comprising a compound according to any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof and one or more pharmaceutically acceptable carriers, diluents or excipients. 5. A pharmaceutical composition according to claim 4, comprising one or more other therapeutic agents. A compound according to any one of claims 1 thereof, for use in therapy. 7. A compound according to any one of claims 1 thereof, for use in reducing weight gain. 8. A compound according to any of claims 1 20 thereof, for use in reducing weight recovery. 9. A compound according to any one of claims 1 thereof, for use in the treatment of obesity. 10. A compound according to any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof, for use in the treatment of type 2 diabetes. to 3, or a pharmaceutically acceptable salt to 3, or a pharmaceutically acceptable salt to 3, or a pharmaceutically acceptable salt to 3, or a pharmaceutically acceptable salt